PROJECT SUMMARY/ABSTRACT The overall goal of this grant over the years has been to describe in molecular details the mechanism of action of physiologically important forms of phospholipase A2 (PLA2). During the course of these studies, it has become apparent that the activity of this superfamily of enzymes depends critically on the interaction of the proteins with large lipid aggregates, where the orientation of the enzyme with respect to the plane of the lipid-water interface can have a dramatic effect on activity. The nature of this interaction has been challenging to explore because it represents the interaction of two large macromolecules. The activity of these enzymes increases when the enzyme is at the lipid-water interface of membranes and micelles. This activation is due in part to changes in enzyme-lipid orientation and to conformational changes in the enzyme. This renewal application will extend our current studies on the human cytosolic Group IVA cPLA2 (cPLA2), the human Ca2+-independent Group VIA iPLA2 (iPLA2), and the human lipoprotein-associated PLA2/PAF (platelet activating factor) acetyl hydrolase Group VIIA LpPLA2 (LpPLA2). The cPLA2 regulates the release of free arachidonic acid for eicosanoid formation critical to inflammatory disease responses. The iPLA2 is responsible for remodeling of membrane phospholipids and plays critical roles in the regulation of mitochondrial phospholipids and several metabolic diseases. The LpPLA2 associates with lipoproteins, both LDL and HDL, where it releases oxidized fatty acids and PAF and is implicated in cardiovascular disease. We will employ amide hydrogen/deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD) simulations to tackle structural questions about how these proteins act that cannot be addressed easily by NMR or X-ray crystallography to determine the interactions of these enzymes with specific substrate phospholipids, large phospholipid vesicles, and specific potent inhibitors. We also will employ our newly developed liquid chromatography MS (LCMS) based LIPID MAPS lipidomics to determine the specificity of each of these enzymes for specific phospholipid polar groups and sn-1/sn-2 acyl chains to establish the functional aspects and will integrate the DXMS/MD structural information with the LCMS specificity information. This work will generate important widely applicable novel information on how several physiologically important phospholipases interact with the lipid-water interfaces of membranes and micelles.